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Extracting Onion DNA
Abstract In this project, you'll learn how to isolate DNA from onion cells, separating it from other cellular components in a manner that still preserves its structure and sequence. In the end, you'll have enough DNA to see with the unaided eye, and you'll be able to spool it to demonstrate its strand-like structure. Objective This procedure is designed to extract DNA from onion in sufficient quantity to be seen and spooled. Materials and Equipment These items can be purchased from Carolina Biological Supply Company, a Science Buddies Approved Supplier: *95% ethanol (2 mL). Note: Make sure to purchase 95% ethanol, not methanol. Use caution when handling ethanol because it is very flammable. Note: If you are ordering this chemical through Carolina Biological Supply Company, the chemical must be ordered by a teacher and shipped to a school or business address, so plan accordingly. *Thermometer that will measure 60°C, such as a candy thermometer *250 mL beaker. Alternatively, a 1-cup measuring cup with mL markings may be used. *1000 mL beakers (2). Alternatively, 4-cup measuring cups may be used. *Funnel. Alternatively, a strainer may be used that will fit in the 1000 mL beaker or 4-cup measuring cup. *Test tube, with cap. Alternatively, a narrow glass container, such as a liqueur glass or clear bud vase can substitute for the test tube. *Optional: Watering pipets. Alternatively, a Pasteur pipet or medicine dropper may be used. You will also need to gather these items: *Freezer *Hot tap water bath, at 55-60°C. A 3-quart saucepan works well to hold the water. *Ice water bath, at 4°C. A large mixing bowl with ice cubes works well. *Measuring spoons *Light-colored dishwashing liquid or shampoo, such as Dawn® or Suave® Daily Clarifying Shampoo *Table salt, either iodized or non-iodized (1/4 tsp.) *Distilled water (90 mL) *Spoons for mixing (4) *Large onion *Food processor, blender, or sharp knife for cutting the onion. *Timer or clock *Optional: Refrigerator #6 coffee filter or cheese cloth (4 layers) *Optional: Flat toothpicks (2) and meat tenderizer that contains papain, such as Adolph's *Glass rod, Pasteur pipette that has been heated at the tip to form a hook, or a similar device. Alternatively, a wooden skewer or nut pick, which is a small metal rod with a curved tip, may also work. *Lab notebook Introduction The process of extracting DNA from a cell is the first step for many laboratory procedures in biotechnology. The scientist must be able to separate DNA from the unwanted substances of the cell gently enough so that the DNA is not broken up. It is both interesting and important to understand the reason for some of the steps in the procedure below. An onion is used because it has a low starch content, which allows the DNA to be seen clearly. The salt shields the negative phosphate ends of DNA, which allows the ends to come closer so the DNA can precipitate out of a cold alcohol solution. The detergent causes the cell membrane to break down by dissolving the lipids and proteins of the cell and disrupting the bonds that hold the cell membrane together. The detergent then forms complexes with these lipids and proteins, causing them to precipitate out of solution. Terms and Concepts Research the following terms before beginning your experiment: *Cell membrane *Lipid *Protein *DNA *Precipitate Experimental Procedure #Put the 95% ethanol in the freezer to chill it. You will use this later, in step 12. #Prepare two water baths—one at 55-60°C and another filled with ice cubes and water, around 4°C. For the hot water bath, a large metal pot can be used along with a thermometer with an appropriate temperature range. For the ice bath, a mixing bowl filled with ice and water works well. #In the 250 mL beaker (or a 1-cup measuring cup), make a solution consisting of 10 milliliters (mL) (1 tablespoon Tbsp.) of liquid dishwashing detergent or shampoo and one level 1/4 teaspoon (1.5 grams) of table salt. #Add distilled water to the beaker to make a final volume of 100 mL. Dissolve the salt by stirring slowly to avoid foaming. #Coarsely chop one large onion with a food processor or blender (may be done by hand if neither is available) and put into a 1000 mL beaker (or a 4-cup measuring cup). For best results, do not chop the onion too finely. The size of the pieces should be like those used in making spaghetti. It is better to have the pieces too large than too small. #Cover the chopped onion with the 100 ml of solution from step 4. ##The liquid detergent causes the cell membrane to break down and dissolves the lipids and proteins of the cell by disrupting the bonds that hold the cell membrane together. The detergent causes lipids and proteins to precipitate out of the solution. The table salt (NaCl) enables nucleic acids to precipitate out of an ethanol solution because it shields the negative phosphate end of DNA, causing the DNA strands to come closer together and coalesce. #Put the 1000 mL beaker with the chopped onion and solution in a hot water bath at 55-60°C, as shown in Figure 1, below, for 10-12 minutes. If you are using a large metal pot for a water bath, remove the pot from the stove before placing the beaker inside—the procedure is safer if the pot is off the burner. Continue to monitor temperature of the water bath and make adjustments as needed (i.e., adding hot or cold water). During this time, also press the chopped onion mixture against the side of the beaker with the back of the spoon. ##Do not keep the mixture in the hot water bath for more than 15 minutes because the DNA will begin to break down. ##The heat treatment softens the phospholipids in the cell membrane and denatures the DNAse enzymes which, if present, would cut the DNA into small fragments so that it could not be extracted. #Cool the mixture in an ice water bath, at about 4°C, as shown in Figure 2, below, for 5 minutes. During this time, press the chopped onion mixture against the side of the beaker with the back of the spoon. This step slows the breakdown of DNA. #Filter the mixture through a #6 coffee filter or four layers of cheese cloth placed in a funnel or strainer over a new 1000 mL beaker (or a 4-cup measuring cup). When you filter the onion mixture, try to keep the foam from getting into the filtrate. It sometimes filters slowly, so you might want to put the whole set up in the refrigerator and let it filter overnight. #Dispense the onion solution into a test tube. The test tube should contain about 5 mL (1 tsp.) of solution or be about 1/3 full. For most uniform results among test tubes, stir the solution frequently when dispensing it into the tubes. There is not an advantage to dispensing more than 5 mL of solution into a test tube. The solution can be stored in a refrigerator for about a day before it is used in the following steps. When the solution is removed from the refrigerator, it should be gently mixed before the test tubes are filled. #Optional: Add two toothpicks full of meat tenderizer to the onion solution, cap the tube, and mix gently to avoid foaming. Meat tenderizer contains papain, an enzyme that will clean extra proteins away from DNA. #Take the cold 95% ethanol out of the freezer and add it to the test tube to create an ethanol layer on top of about 1 centimeter (cm). For best results, the ethanol should be as cold as possible. The ethanol can be added to the solution in at least three ways:DNA is not soluble in ethanol. When ethanol is added to the mixture, all the components of the mixture, except for DNA, stay in solution while the DNA precipitates out into the ethanol layer. ##Fill a watering pipet, pasteur pipette, or medicine dropper with ethanol, put it to bottom of the test tube, and release the ethanol. ##Or, put about 1 cm of ethanol into the bottom of a test tube and add the onion solution. ##Or, slowly pour the ethanol down the inside of the test tube with a watering pipet, pasteur pipette, or medicine dropper. #Let the solution sit for 2-3 minutes without disturbing it. It is important NOT to shake the test tube. You can watch the white DNA precipitate out into the ethanol layer. When good results are obtained, there will be enough DNA to spool on to a glass rod, a pasteur pipette that has been heated at the tip to form a hook, or similar device. A wooden skewer or nut pick (small metal rod with curved tip) may also work well for spooling DNA if Pasteur pipette is unavailable. DNA has the appearance of white mucus. Photos Category:Biotechnology Experiments